The present invention relates to recombinant virus strains capable of killing tumor cells. More specifically, the present invention relates to a mutated replication-competent viruses which contains mutations in two genes, is hypersensitive to antiviral agents such as ganciclovir, is not neurovirulent and does not replicate in non-dividing cells, yet can kill nervous system tumor cells. The present invention also relates to recombinant herpesvirus strains, vital vaccines incorporating such strains, methods for making such strains and vaccines, and methods for immunizing a human host against herpes simplex virus using the vaccines.
Malignant tumors of the nervous system are generally fatal, despite many recent advances in neurosurgical techniques, chemotherapy and radiotherapy. In particular, there is no standard therapeutic modality that has substantially changed the prognosis for patients diagnosed with malignant brain tumors. For example, high mortality rates persist in malignant medulloblastomas, malignant meningiomas and neurofibrosarcomas, as well as in malignant gliomas.
Gliomas are the most common primary tumors arising in the human brain. The most malignant glioma, the glioblastoma, represents 29% of all primary brain tumors, some 5,000 new cases per year in the United States alone. Glioblastomas are almost always fatal, with a median survival of less than a year and a 5-year survival of 5.5% or less. Mahaley et al., J. Neurosurg. 71: 826 (1989); Shapiro, et al., J. Neurosurg. 71: 1 (1989): Kim et al., J. Neurosurg. 74: 27 (1991). After glioblastomas are treated with radiotherapy, recurrent disease usually occurs locally; systemic metastases are rare. Hochberg et al., Neurology 30: 907 (1980). Neurologic dysfunction and death in an individual with glioblastoma is due to the local growth of the tumor.
Efforts to cure primary and metastatic brain tumors have focused on new approaches that make use of genetically modified viruses either to deliver cytotoxic genes to tumor cells or to directly infect and destroy tumor cells in a selective fashion. Treatment strategies employing replication-competent HSV-1 mutants may be particularly promising (Hum. Gene Ther. 5, 183-191; Cancer Res. 54, 5745-5751.; J Neuro-Oncol, 19, 137-147: J. Neurosurg, 77, 590-594: Neurosurg. 32, 597-602; Science 252, 854-856; Stereotact. Funct. Neurosurg. 59, 92-99; Nature Med. 1, 938-943; Virol. 211, 94-101.) Such mutants, like wild-type HSV-1 strains, establish a lytic infection in dividing tumor cells, leading to tumor cell destruction, but establish only a latent infection of the surrounding nondividing brain cells, including neurons. These mutants are attenuated human pathogens and thus, must be examined fully for their safety and utility prior to clinical use.
The first HSV-1 mutant studied, dlsptk, carried a single mutation in the thymidine kinase (TK) gene. Mutant strain dlsptk was found to have significant antineoplastic efficacy with a minimal level of toxicity in human tumor xenografts in immunodeficient mice (Neurosurg. 32, 597-602; Science 252, 854-856). These effects demonstrated the potential of HSV-1 as a tumor therapy, but at least two concerns regarding the safety of dlsptk limited its potential for human use. Because it lacks a functional TK gene, strain dlsptk cannot be controlled by the antiherpetic drugs acyclovir or ganciclovir. Second, infection with TK mutant strains causes neurovirulence in animal models when used at doses that would be employed for cancer therapy (N. Engl. J. Med. 320, 293-296). For these reasons, investigators have tested the usefulness of other mutations that severely reduce the ability of the virus to replicate in nondividing cells but do not prevent viral replication in actively dividing cells (Cancer Res. 54, 5745-5751: J Neuro-Oncol. 19, 137-147; J. Neurosurg. 77, 590-594; Neurosurg. 32, 597-602; Virol. 211, 94-101). One such mutation, introduced in both copies of the diploid ICP34.5 gene, has been shown in at least two parental HSV-1 backgrounds to result in viral vectors that have the tumor kill efficiency of dlsptk but minimal to no detectable toxicity (J. Neurosurg. 77, 590-594: Neurosurg. 32, 597-602; Virol. 211, 94-101. J. Gen. Virol. 75, 2059-2063.). Importantly, the HSV-1 strains with mutation of only the ICP34.5 genes retain TK activity, allowing for control by antiherpetic drugs that are activated by HSV-encoded TK. In U.S. Pat. No. 5,328,688. Roizman. issued Jul. 12, 1994, there is disclosed an HSV-1 strain that is reported to be rendered avirulent by the prevention of expression of an active product of a gene, designated gamma 34.5. that maps to the inverted repeats, flanking the long unique sequence of herpes simplex virus DNA. this gene is not essential for viral growth in cell culture. Viruses from which 34.5 was deleted or which carried premature stop codons in the 34.5 gene are avirulent following intracerebral inoculation of mice.
More recently, efforts to increase the safety of herpes-based therapy have spurred the development of HSV-1 strains that have mutations in two viral genes and thus are theoretically less likely to be repaired by recombination with a preexisting or subsequent HSV-1 infection. U.S. Pat. No. 5,585,096, Martuza et al., issued Dec. 17, 1996, discloses a method for killing malignant brain tumor cells in vivo by introducing replication-competent herpes simplex virus vectors to tumor cells. A replication-competent herpes simplex virus vector, with defective expression of the gamma 34.5 gene and the ribonucleotide reductase gene, specifically destroys tumor cells and is not neurovirulent.
Initial success with such a strain, designated G207, has been reported for treatment of glioblastoma xenografts established in immunodeficient mice (Nature Med. 1, 938-943). Strain G207 carries a deletion of both copies of the ICP34.5 gene and a mutated ICP6 gene, that encodes the large subunit of the ribonucleotide reductase, an enzyme in the salvage pathway required for efficient DNA synthesis (ROIZMAN, B. and SEARS. A. E., 1990). Herpes simplex viruses and their replication, p. 1795-1841. In B. N. Fields, et al. (ed.), Virology, 2nd ed. Raven Press, New York). In addition to being multiply-mutated, this virus also retains sensitivity to antiherpetic drugs and has minimal toxicity in animal models (Nature Med. 1, 938-943).
It remains of utmost importance to develop a multiple HSV-1 mutant viral strain that has the greatest possible safety and therapeutic value.
It is therefore an object of this invention to provide a replication-competent viral vector, suitable for use in humans, that is capable of killing human tumor cells in vivo, that exhibits hypersensitivity to anti-viral agents and an inability to revert to wild-type virus, and that is not neurovirulent at a dose required to kill tumor cells.
It is another object of the present invention to provide for the production of a replication-competent herpes simplex virus-derived vector that is effective and safe for use in the treatment of malignant brain tumors in humans.
It is a further object of the invention to provide a safe, mutated HSV-1 vector, incapable of reverting to wild-type form through a spontaneous single mutation, for use in the context of a vaccine or tumor therapy.
Still another object of the present invention is to provide a mutant HSV-1 vector that can selectively replicate in and kill a tumor cell of non-nervous tissue origin.
An additional object of the present invention is the production of a replication-competent viral vector, derived from herpes simplex virus, that can be employed in a genetic therapy against tumors by expressing foreign genes to target an immune response that kills the tumor cells.
Yet another object of the present invention is the production of a mutant herpes simplex virus vector containing a tumor cell-specific promoter so that the vector can be targeted to specific tumor cells.
It is also an object of the present invention to provide for production of a replication competent viral vector that is effective and safe for use as a vaccine to protect against infection by herpes simplex virus.
In satisfying these and other objects, there has been provided, in accordance with one aspect of the present invention, a replication-competent herpes simplex virus that is incapable of expressing both (i) a functional gamma 34.5 gene product and (ii) a uracil DNA glycosylase. In a preferred embodiment, the vector contains alterations in both genes.
In accordance with another aspect of the present invention, a method has been provided for killing tumor cells in a subject, comprising the step of administering to the subject a pharmaceutical composition comprising (A) a herpes simplex virus vector that is altered in (i) the gamma 34.5 gene, and (ii) the a uracil DNA glycosylase (UNG) gene; and (B) a pharmaceutically acceptable vehicle for the vector, such that the tumor cells are altered in situ by the vector and the tumor cells are killed. The tumor cells can be of a nervous-system type selected from the group consisting of astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma, Schwannoma, neurofibrosarcoma, and medulloblastoma. Other kinds of tumor cells which can be killed, pursuant to the present invention, include those selected from the group consisting of melanoma cells, pancreatic cancer cells, prostate carcinoma cells, breast cancer cells, lung cancer cells, colon cancer cells lymphoma cells, hepatoma cells and mesothelioma and epidermoid carcinoma cells.
In accordance with still another aspect of the present invention, a method is provided for killing tumor cells in a subject, comprising the steps of administering to the subject a herpes simplex virus vector, wherein the vector comprises a tumor cell-specific promoter wherein the promoter controls expression of at least one viral protein necessary for viral replication and wherein the promoter is induced selectively or at a higher level in tumor cells than in normal cells. This method can entail the use of a promoter that is selectively capable of expression in nervous-system tumor cells, for example, glioblastoma cells, medulloblastoma cells, meningioma cells, neurofibrosarcoma cells, astrocytoma cells, oligodendroglioma cells, neurofibroma cells, ependymoma cells and Schwannoma cells.
A method also is provided for preparing a replication-competent vector of a herpes simplex virus, comprising the steps of (A) isolating a viral genome of the herpes simplex virus: and (B) permanently altering the genome so that the virus is (1) sensitive to antiviral agents, (2) kills tumor cells and (3) expresses decreased generalized neurovirulence. For example, the vector can be derived from either HSV-1 or HSV-2.
The present invention further provides for a method of protecting a subject against herpes simplex virus infection, comprising the step of administering to the subject a pharmaceutical composition that is comprised of (A) a herpes simplex virus vector wherein the genome of the virus is altered in (i) the gamma 34.5 gene, and (ii) the a uracil DNA glycosylase gene; and (B) a pharmaceutically acceptable vehicle for the vector.
According to still another aspect of the present invention, there has been provided a method of eliciting an immune response to a tumor cell, comprising the step of administering to the subject a pharmaceutical composition comprising (A) a herpes simplex virus, wherein the genome of the virus (i) contains an expressible non-herpes simplex virus nucleotide sequence encoding a desired protein capable of eliciting an immune response in the subject, and (ii) is altered in the gamma 34.5 gene, and the uracil DNA glycosylase gene: and (B) a pharmaceutically acceptable vehicle for the virus. In a preferred embodiment, the method further comprises the step of co-administration with neurosurgery, chemotherapy or radiotherapy.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.